1. Field of the Invention
The invention relates to methods for purification and manipulation of hemopoietic progenitor cells and lymphocytes. More particularly, the invention relates to methods for identifying, manipulating and purifying pluripotent hemopoietic stem cells (PHSC) and certain lymphocytic types to provide or increase populations of such cells for a variety of therapeutic and research purposes.
2. Summary of the Related Art
Pluripotent hemopoietic stem cells (PHSC) comprise less than about 1% of total bone marrow cells, and are self-renewing cells that give rise to all the different types of blood cells. Research directed toward isolation and characterization of PHSC has a history of over 20 years.
Visser et al., Exp. Hematol. 18: 248-256 (1990), reviews research concerning purification of PHSC, including partial purification strategies used to avoid graft-versus-host disease in heterologous bone marrow transplantation and to remove malignant cells from bone marrow for autologous transplantation.
Various methods have been developed to assay PHSC. For example, Sutherland et al., Proc. Natl. Acad. Sci. USA 87: 3584-3588 (1990), discloses an assay ("long term culture (LTC) initiating cell assay") for PHSC using measurement of ability to give rise to short-term colony forming cells five to eight weeks after overlay on an irradiated stromal layer.
Methods of purifying PHSC based on immunological techniques involving surface antigens are known in the art. Sutherland, et al., Blood 74: 1563-1570 (1990), discloses an immunological method which provides about 800-fold enrichment of PHSC and utilizes, inter alia the fact that PHSC express the CD34 antigen, which is otherwise expressed only by clonogenic cells at a lower level.
Purification of PHSC based on differential rhodamine 123 (Rh) accumulation is also known in the art. Bartoncello et al., Exp. Hematol. 13: 999-1006 (1985), discloses a method for enriching PHSC, using fluorescence-activated cell sorting (FACS) to separate the Rh-dull PHSC from the Rh-bright remaining cells. Srour et al., Exp. Hematol. 18: 549 (1990) teaches that PHSC are an Rh-dull subset of CD34-expressing cells.
Reduced accumulation of Rh has been attributed to the expression of P-glycoprotein, a multidrug transporter, in some cell types, but has been attributed to reduced numbers or activity of mitochondria in PHSC.
Neyfakh et al., Exp. Cell Res. 174: 168-176 (1988), discloses that multidrug resistant hamster fibroblasts accumulate lower concentrations of fluorescent dyes, including Rh, than do their drug-sensitive counterparts, and concludes that this difference results from increased efflux of fluorescent dyes from the multi drug-resistant cells. Neyfakh et al., Exp. Cell. Res. 185: 496-505 (1989), teaches that multidrug resistance in B-lymphoma cell lines and T-lymphocytes is inversely proportional to accumulation of fluorescent dyes, including Rh, that there are normal mouse T-lymphocytes that demonstrate rapid efflux of Rh which can be prevented by inhibitors of the multidrug transporter system, and that an Rh-effluxing T-lymphoma contained high levels of P-glycoprotein mRNA. In contrast, McCarthy et al., I. J. Cell Cloning 8: 184-195 (1990), teaches that decreased Rh accumulation in PHSC results from a reduced number of Rh-accumulating mitochondria in these quiescent cells. See also Spangrude et al., Proc. Natl. Acad. Sci. USA 87: 7433-7437 (1990).
Thus there are currently at least two available methods for enriching for PHSC. Each of these available methods has limitations, however. Enrichment based on CD34 recognition by antibodies cannot fully purify PHSC, since other cells also express CD34. Enrichment based on FACS separation of rhodamine-dull cells also imperfectly purifies PHSC and is not readily amenable to multi-parameter FACS analysis due to the overlap between the fluorescent signal produced by rhodamine and those produced by other probes.
There is, therefore, a need for additional methods for purifying PHSC which may be separately used, in conjunction with each other, or in conjunction with existing methods to effect better purification of PHSC than is presently available. Such methods may be based upon antibody recognition or upon differential accumulation of molecules between PHSC and other cell types.